Inhibition of Soybean 15-Lipoxygenase and Human 5-Lipoxygenase by Extracts of Leaves, Stem Bark, Phenols and Catechols Isolated From Lithraea caustica (Anacardiaceae)

Lithraea caustica (Molina) Hook. and Arn. (Anacardiaceae), common name Litre, is an evergreen endemic plant used in the Mapuche Chilean folk medicine. The stem juice of L. caustica mixed with Rubus ulmifolius (blackberry) is used to treat cough and the infusion of leaves is used in baths to treat joint inflammations. In this study, the activities of 3-n-alk(en)yl-catechols, obtained from the dichloromethane extract of the epicuticular compounds of fresh leaves (DCME), stem bark petroleum ether extract (PEE), fractions of phenols and phenol-acid compounds obtained from the methanolic extract (methanolic extract) of defatted leaves and aqueous infusion (AE) from fresh leaves, were evaluated as in vitro inhibitors of soybean 15-lipoxygenase (15-sLOX) and human 5-lipoxygenase (5-hLOX), one of the inflammation pathways. The 3-n-alk(en)yl-catechols were characterized by gas chromatography-mass spectrometry and 1D and 2D nuclear magnetic resonance analysis as mixtures of 3-[(10E)-pentadec-10′-en-1-yl]-catechol, 3-[(10Z)-pentadec-10′-en-1-yl]-catechol and 3-n-pentadecylcatechol. In addition, two fractions, obtained from MeOHE, were characterized by liquid chromatography electrospray ionization tandem mass spectrometric as complex mixtures of known acids and phenolic compounds. DCME, MeOHE and ethyl acetate extract (AcOEtE) extracts showed inhibition against 15-sLOX, and the AE of fresh leaves, showed the best inhibition against 5-hLOX. The mixture of 3-n-alk(en)yl-catechols showed inhibition of 15-sLOX and 5-hLOX. The compounds 3-[(10Z)-pentadec-10′-en-1-yl]-catechol (IC50 2.09 µM) and 3-n-pentadecylcatechol (IC50 2.74 µM) showed inhibition against 5-hLOX. The inhibition values obtained for the 3-n-alk(en)yl-catechols are in the range of well-known inhibitors of 5-hLOX. Acetylation of the 3-n-alk(en)yl-catechols blocks the inhibitory activity, indicating that the free catechol function is necessary for the enzyme inhibition. In addition, the fractions of phenols and phenol-acid compounds showed inhibitory activity against 15-sLOX and the AE, showed a good inhibition against 5-hLOX. These results would be in agreement with the use of L. caustica, as an anti-inflammatory in Mapuche ethnomedicine.

To validate, with scientific evidence, the anti-inflammatory use of L. caustica in Mapuche ethnomedicine, a phytochemical study of the leaves and stem bark of L. caustica was carried out. The different extracts, mixture of compounds and pure compounds were analyzed as inhibitors of 5 human lipoxygenase (5-hLOX) and 15 soybean lipoxygenase . 15-sLOX and 5-hLOX, are enzymes that use molecular oxygen in the dioxygenation of arachidonic acid (AA) to form hydroperoxides (Boyington et al., 1993;Saura and Jean-Didier, 2016;Snodgrass and Brüne, 2019) from 1,4-diene units (Chohany et al., 2011) and which are related in the biosynthesis of lipoxins (LXs) and leukotrienes (LTs) (Vásquez-Martínez et al., 2019). They play a role in the pathogenesis of inflammatory, hyperproliferative, neurological, and metabolic diseases (Dobrian et al., 2011). It is important to mention, that 15-sLOX is used as model of 5-hLOX due to their structural similarity and mechanism of action (Wecksler et al., 2009).

Preparation of Crude Extracts, Fractionation, Isolation and Characterization of Components
Extracts were obtained following the methodology described by Huanquilef et al., (2020) with some modifications.

Petroleum Ether Extract From Stem Bark
Dried and milled stem bark of L. caustica (290 g) was extracted in Soxhlet apparatus for 8 h using 2.5 L of petroleum ether. The extract was dried over anhydrous sodium sulphate and filtered using a fritted glass funnel. The solvent was evaporated under reduced pressure in a rotatory evaporator, obtaining PEE (1.6 g, 0.55%) (Figure 1).

Dichloromethane Extract From Leaves
Leaves of L. caustica (1.4 kg), were extracted by dipping the fresh plant material in 5 L of cold CH 2 Cl 2 for 5 min, for the extraction of the epicuticular components (Urzúa et al., 2011). The extract was dried over anhydrous sodium sulphate and filtered using a fritted glass funnel. The solvent was evaporated under reduced pressure in a rotatory evaporator obtaining DCME (6.1 g, 0.45% from fresh plant material) (Figure 1).

Ethyl Acetate Extract From Methanolic Extract
Dried defatted milled leaves of L. caustica, previously extracted with DCM (50 g), were extracted with 2 L MeOH using a Soxhlet apparatus for 4 h and the solvent was evaporated under reduced pressure in a rotatory evaporator, obtaining the MeOHE (14 g, 28% from plant material). The syrupy MeOHE was dissolved with 150 ml of H 2 O at 40°C, allowed to stand at room temperature and the suspension filtered using a fritted glass funnel. The solid was discarded and the filtrate extracted by liquid-liquid extraction with ethyl acetate (AcOEt) (4 × 30 ml). The solvent was evaporated under reduced pressure in a rotatory evaporator, obtaining AcOEtE (1.0 g, 2.0% from MeOHE) (Figure 1).

Fractionation of Ethyl Acetate Extract
The AcOEtE was re-suspended in 50 ml of AcOEt at 60°C and allowed to stand at room temperature. The suspension filtered using a fritted glass funnel. The solid was discarded and the filtrate extracted with 5% sodium bicarbonate (3 × 30 ml). The organic layer was dried over anhydrous sodium sulphate, filtered using a frit funnel and the solvent was evaporated under reduced pressure in a rotatory evaporator, obtaining AcOEtE-1 (140 mg, 14%, from AcOEtE; phenolic compounds fraction). The basic extract, kept at 0°C, was stirred and neutralized dropwise with concentrated hydrochloric acid, and extracted with AcOEt (4 × 30 ml). The AcOEt extract was dried over anhydrous sodium sulphate, filtered using a frit funnel and evaporated under reduced pressure in a rotatory evaporator, obtaining AcOEtE-2 (400 mg, 40%, from AcOEtE; acid-phenolic compounds fraction) ( Figure 1). The AcOEtE-1 (phenolic compounds fraction) and AcOEtE-2 (acidphenolic compounds fraction) were analyzed by LC-ESI-MS/MS.

Aqueous Extract From Leaves
Fresh leaves of L. caustica (314 g) were extracted to obtain an infusion, by dipping the fresh plant material in 700 ml of hot distilled water at 80°C for 5 min. The extract was filtered using a fritted glass funnel and the solvent was evaporated under reduced pressure in a rotatory evaporator obtaining AE (3.1 g, 0.98% from fresh plant material) ( Figure 1).
Fractions with similar chromatograms were combined and further purified by column chromatography, to produce 193 mg of a phenol fraction (catechols) from the DCME and 140 mg of a phenol fraction (catechols) from the PEE. Purity of the fractions and composition was obtained through thin-layer chromatography, GC-MS, FTIR and nuclear magnetic resonance (NMR) analysis.

Gas Chromatography-Mass Spectrometry Analysis
The analysis were performed in a gas chromatograph Shimadzu model GC-MS-QP 2010 Ultra (Shimadzu, Kyoto, Japan), operating in the splitless mode and fitted with a capillary GC column Rtx-5MS cross bond 5% diphenyl -95% dimethyl polysiloxane (30 m length, 0.25 mm I.D., 0.25 µm film thickness) (Restek, Bellefonte, PA, United States). Analysis by GC-MS of the catechol fractions was done using the following conditions, column temperature was held at 40°C for 5 min, raised at 10°C/min to 200°C and maintained for 5 min and the column temperature was raised at 3°C/min to 290°C and maintained for 20 min at 290°C. The injection volume was 1 µl and the carrier gas was helium (flow rate: 1.3 ml/min). The mass spectrometer was used in the electron impact ionization mode (70 eV) with an emission current of 250 µA and acquisition mass range, 50-500 Dalton. The temperatures of the injection port, ion source and transfer line were 250, 240 and 260°C, respectively. The instrument was operated in the scan mode. In the scan mode, the instrument monitors a wide and continuous range of masses determined by the molecular masses and fragmentation patterns of the potential compounds of interest. The identification of compounds in the chromatographic profiles were achieved by comparison of the compounds fragmentation with data from the literature.

Fourier Transform Infrared Spectroscopy Analysis
The samples were analyzed by Fourier transform infrared spectroscopy (FTIR), on a Bruker 66v Fourier-transform infrared spectroscopy spectrometer (4,000-400 cm −1 ). The samples were dissolved in methylene chloride and were analyzed in film.

High Performance Liquid Chromatography-Diode-Array-Detector Analysis
High performance liquid chromatography-diode-array-detector (HPLC-DAD) analysis were performed using liquid chromatograph (Waters 600; Milford, MA, United States) with a reverse-phase Symmetry Shield RP18 column (5-µm particle size; 25 × 0.46 cm). Gradient elution was performed using a mobile phase of 0.1% acetic acid in water (solution A) and 0.1% Frontiers in Pharmacology | www.frontiersin.org November 2020 | Volume 11 | Article 594257 acetic acid in acetonitrile (solution B): 0-5 min, isocratic elution with 70% A/30% B; 5-45 min, linear gradient from 70 A/30 B to 55% A/45% B. A Waters 2996 DAD was used to detect the compounds and their spectra were recorded at wavelengths of 200-800 nm. Quantification was based on the areas of the peaks in the chromatograms, which were determined at 254 nm.

Nuclear Magnetic ResonanceAnalysis
Mono-dimensional 1 H, 13 C and DEPT-135, bi-dimensional homonuclear COSY, and heteronuclear bi-dimensional HSQC-ed and HMBC NMR spectrum were obtained on a Bruker DPX 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C). Samples were dissolved in CDCl 3 , and the spectra were calibrated using TMS signals. The chemical shifts are given in ppm.

Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric Analysis
The LC-ESI-MS/MS analysis were performed using a LC-ESI-MS/MS system consisted in a HPLC HP1100 (Agilent Technologies Inc., CA-United States) connected to the mass spectrometer Esquire 4000 Ion Trap LC/MS (n) system (Bruker Daltonik GmbH, Germany). A column Kromasil 100-5C18 of 250 × 4.6 mm, 5 μm and 100Å (Eka Chemicals AB, Sweden) was used for the analysis; at the exit of the column a split divided the eluent for simultaneous UV spectroscopy detection and mass spectrometry detection. The mobile phase was formic acid in water (0.1% v/v, solvent A) and formic acid in acetonitrile (0.1% v/v, solvent B) at a flow rate of 1 ml/min according to the following elution gradient: 0-5 min, 10% B; 5-20 min, 10-30% B; 20-52 min, 30-45% B; 52-53 min, 45-10% B and 53-60 min, 10% B. Compounds were detected at 254 nm. The mass spectral data were acquired in positive and negative modes; ionization was performed at 3,000 V assisted by nitrogen as nebulizing gas at 24 psi and as drying gas at 365°C and a flow rate of 6 L/min. All scans were performed in the range m/z 20-2,200. The trap parameters were set in ion charge control using manufacturer default parameters. Collision induced dissociation (CID) was performed by collisions with the helium background gas present in the trap and automatically controlled through Smart Defrag option. The tentative identification of the compounds in each fraction were based on: i) comparison of experimental fragmentation vs. library or literature fragmentation.; and ii) correlation between both polarities (however, some compounds were only observed in one ionization mode) and adduct presence. Those compounds observed in both polarities were labeled with M + H, M + Na or M−H.
Samples of 3-n-alk(en)yl-catechols mixture of the phenolic fraction of L. caustica stem bark (25 mg) and 3-[(10Z)pentadec-10′-en-1-yl]-catechol (2) (25 mg) and Pd/C (5 mg) in CH 2 Cl 2 (2.5 ml) were respectively stirred under H 2 at room temperature for 24 h. The reaction mixture was filtered, using a fritted glass funnel with Celite. The solvent was evaporated under reduced pressure in a rotatory evaporator, yielding the corresponding reduction products. The product of each reactions was purified by column chromatography and analyzed by GC-MS, FTIR and 1 H and 13 C NMR and identified in each reaction as 3-n-pentadecylcatechol (3) (Muñoz-Ramírez et al., 2020).

Evaluation of Bioactivity
In Vitro Assay of 15 Soybean Lipoxygenase Inhibition Assays of inhibition of 15-sLOX were performed using a previous published methodology (Tirapegui et al., 2017), with minor modifications. In shortly, the activity of 15-sLOX (Cayman Chemical Item No. 60712) was determined following the formation of reaction products at 234 nm (ε 25,000 M −1 cm −1 ) with a Perkin-Elmer Lambda 25 UV/Vis (LabMakelaar Benelux B.V. Zuid-Holland, Nederland). All reactions were performed at a final volume of 2 ml and stirred using a magnetic bar at room temperature. The reaction medium used contained 0.1 M HEPES buffer (pH 7.4), Triton X-100 0.01%, and the linoleic acid substrate at a 10 μM concentration determine preliminary percent inhibition (% I). The concentration was determined quantitatively by allowing the enzymatic reaction to go to completion. The reaction was carried out by adding the inhibitor (sample) in methanol to the cuvette with the substrate buffer, and finally the enzyme was added. Nordihydroguaiaretic acid was used as positive control. Assays were performed in duplicate on two different days. IC 50 values were determined by measuring enzyme activity at different concentrations of inhibitor dissolved in methanol; in the range of the initial velocity of the enzyme reaction. Finally, the inhibition% vs. the inhibitor concentration was plotted, giving a hyperbolic saturation curve using GraphPad Prism Demo.

In Vitro Assay of 5 Human Lipoxygenase Inhibition
Assays of inhibition of 5-hLOX were performed using a previous published methodology (

RESULTS AND DISCUSSION
Analysis of the Catechols Fraction from the Petroleum Ether Extract From Stem Bark Litharea Caustica by Gas Chromatography-Mass Spectrometry fraction of phenolic compounds from PEE, analyzed by GC-MS, correspond to a mixture of three compounds ( Figure 2) and compounds 1 and 2 showed identical mass spectra, with molecular ion peaks at m/z 318, consistent with the molecular formula C 21 H 34 O 2 (five unsaturations) and a base peak at m/z 123 (C 7 H 7 O 2 ), consistent with a di-hydroxylated tropylium ion ( Figure 3). The base peak of these spectra was coincident with the spectrum of 3-[(10Z)-pentadec-10′-en-1-yl]-catechol (2) previously isolated from L. caustica stem bark (Gambaro et al., 1986). The mass spectra of compound (3) exhibit a molecular ion peak at m/z 320 consistent with the molecular formula C 21 H 36 O 2 (four unsaturations) and two intense peaks at m/z 123 and 124 (C 7 H 7 O 2 and C 7 H 8 O 2 , respectively), consistent with a dihydroxylated tropylium ion. The main peaks of these spectra were coincident with previously reported spectrum for 3-npentadecycatechol (3) (Gross et al., 1975;Alé et al., 1997). To confirm the presence of compounds (2) and (3), the mixture 3-n-alk(en)yl catechols of the PEE, was analyzed by GC, using 3-[(10Z)-pentadec-10′-en-1-yl]-catecol (2) and 3pentadecylcatecol (3) as standards. Observing the overlap of the chromatograms obtained for each sample analyzed (see Supplementary Figure S8 in the Supplementary Material).

Characterization of 3-[(10E)-Pentadec-109en-1-yl]-Catechol (1)
Compound (1) shows EIMS m/z: 318 (M + ) consistent with the molecular formula C 21 H 34 O 2 (five unsaturations) and a base peak at m/z 123 consistent with the formula C 7 H 7 O 2 (di-hydroxylated tropylium ion). The 3-n-alk(en)yl-catechols mixture was subjected to NMR analysis. The 13 C NMR spectra showed in the aromatic region, the same signals of compound (3) and (2) with only small differences in the chemical shifts. Two signals at δ 129.92 and 129.88 ppm, were assigned to C-10′ and C-11′; C-9′ and C-12′ at δ 29.8 and 30.0 [see Supplementary Figure S4A,B in Supplementary  Material]. E stereochemistry of the double bond was obtained by correlation with δ values in the 13 C NMR spectra of Z and E isomers of 9-tetradecene-1-yl-acetate (Rossi, 1982).

Compounds Characterized in the Ethyl Acetate Extract-1 (Phenolic Compounds) and Ethyl Acetate Extract-2 (Acid-Phenolic Compounds) Fractions From Ethyl Acetate Extract
The fractions AcOEtE-1 and AcOEtE-2, were analyzed in positive and negative mode, by LC-ESI-MS/MS. Results are showed in Tables 1, 2. Table 1 contains the precursor m/z and fragmentations obtained in positive and negative polarity for the 28 chromatographic UV peaks detected for AcOEtE-2 (see Supplementary Figure S5   and 7-6″. Due to the complexity of extract, co-elution was observed for various peaks, this was elucidated with further examination of the fragmentation; as in the cases of peak 5 finally identified as trigalloyl hexose, peak 7 identified as HHDP-galloyl hexose and peak 20 assigned as quercetin-O-galloyl hexoside. Other compounds were identified by comparison with literature data as for example hydroxycinnamic acid-galloyl hexoside in peak 14, pentagalloyl hexose in peak 16, malic acid-digalloyl hexose in peak 21, myricetin-O-acetyl rhamnoside and myricetin-O-galloyl rhamnoside in peak 26. Peak 27 showed a m/z 505 signal with a fragmentation pattern different from that observed for signal m/z 505 from peak 23, a m/z 329 fragment probably due to loss of glucuronide residue, the fragments m/z 329, 316 and 301 probably formed by the successive loss of methyl groups in addition to the characteristic quercetin fragments m/z 151 and 179 suggesting based on the literature of the presence of quercetin-dimethyl ether-Oglucuronide (Falcão et al., 2013). The differentiation of luteolin or kaempferol derivatives was based on their characteristic fragments such as m/z 199 and 175 in the negative mode fragmentation of luteolin (Sánchez-Rabaneda et al., 2003;Sánchez-Rabaneda et al., 2004) or m/z 165 and 121 in the positive fragmentation of kaempferol (Cuyckens and Claeys, 2004;Justino et al., 2009). Other peaks were not characterized. Table 2 contains the precursor m/z signals and fragmentations obtained in positive and negative polarity for the 25 chromatographic UV peaks detected for AcOEtE-1 extract (see Supplementary Figure S6 in Supplementary Material), the tentative identifications were based on the same parameters explained for the Table 1. Some identifications are repeated which is probably due to the presence of isomers as for trigalloyl hexose detected in peaks 1 and 2 and for the compounds quercetin-O-galloyl hexoside (peaks 3 and 9), quercetin-O-rhamnoside (peaks 7 and 9), kaempferol-Orhamnosyl hexoside (peaks 18 and 20) where the isomers are mainly due to the different position of sugars moiety. Peaks 20, 22 and 23 showed the presence of biapigenin-type biflavones based on their fragmentation would correspond to amentoflavone, cuppressuflavone or other which differentiate by their apigenin inter-linkage. Co-elution was observed for various peaks which would be due to the complexity of extract. In some cases, the identification required a further examination of the fragmentation due to differences between experimental and library fragmentation as in the cases of peak 2 finally identified as trigalloyl hexose, of peaks 9 and 12 assigned as quercetin-O-galloyl hexoside. Other compounds were identified by comparison with literature data as for example quercetin-Oacetyl hexoside in peak 13, isorhamnetin-O-rhamnoside and quercetin-dimethyl ether-O-glucuronide in peak 14, myricetin-O-rhamnosyl hexoside in peak 15. For peak 4 was observed in positive polarity a signal m/z 601 with a fragment m/z 287 assigned as kaempferol, a fragment m/z 430 that would be due to the loss of gallic acid (171 Da), a low intensity fragment m/z 437 which would be due to the loss of an hexoside residue suggests the presence of a kaempferol galloyl derivative probably kaempferol-O-galloyl hexoside. Peak 13 showed in positive polarity the signal m/z 507 identified as quercetin-Oacetyl hexoside based on the similarity of its fragmentation with that of delphinidin-O-acetyl hexoside (Favretto and Flamini, 2000;Brito et al., 2014) however it showed the characteristic fragmentation of quercetin and confirmed the identification in negative polarity. Peaks 17 and 18 presented in negative polarity a signal m/z 624 that showed the same fragmentation pattern, a fragment m/z 315 identified as isorhamnetin, a fragment m/z 471 that would be formed by the loss of a gallic acid residue (152 Da), the data suggest the presence of an isorhamnetin galloyl derivative. Peak 19 was identified as quercetin-O-succinyl rhamnoside mainly based on the similarity of its fragmentation with that of peonidin-O-succinyl rhamnoside (De Brito et al., 2007) but it showed the characteristic fragmentation of quercetin. The differentiation of luteolin or kaempferol derivatives was based on their characteristic fragments such as m/z 199 and 175 in the negative fragmentation of luteolin (Sánchez-Rabaneda et al., 2003;Sánchez-Rabaneda et al., 2004) or m/z 165 and 121 in the positive fragmentation of kaempferol (Cuyckens and Claeys, 2004;Justino et al., 2009). Other peaks were not characterized. In Vitro Assay of 15 Soybean Lipoxygenase and 5 Human Lipoxygenase Inhibition The Table 3 shows the IC 50 values against 15-sLOX and 5-hLOX. The extracts were evaluated on 15-sLOX and only AE was tested against 5-hLOX. The pure catechols and catechol mixture were tested against 15-sLOX and 5-hLOX. (see Supplementary Figure  S7A-D in Supplementary Material).
Regarding the activity of the extracts of L. caustica, the DCME was the most potent inhibitor for 15-sLOX and the activity was correlated with the presence of 3-[(10Z)-pentadec-10′-en-1-yl]catechol (2) in the leaves cuticle (Urzúa et al., 2011). The inhibition values (µg/ml) against 15-sLOX produced by extracts of L. caustica were comparable with active extracts from other plant species (Chung et al., 2009). In the sub-extracts, AcOEtE-1 and AcOEtE-2, obtained by fractionation of the AcOEtE (Figure 1.), phenols and phenol acids were respectively identified (Tables 1, 2). Several of the identified compounds have shown anti-inflammatory activity via LOX.

CONCLUSION
The epicuticular DCME showed high inhibitory activity against 15-sLOX and 5-hLOX and showed more selectivity against 5h-LOX, the activity was correlated with the presence of 3-[(10Z)-pentadec-10′en-1-yl]-catechol (2) which also present inhibition of both enzymes isoforms. The AcOEtE, obtained from the MEOHE showed inhibition of 15-sLOX (IC 50 70.69 mg/L). The fractionation of AcOEtE showed two sub-fractions with greater activity. Analysis by HPLC-DAD and LC-ESI-MS/MS, confirmed a complex mixture of phenolic compounds in AcOEtE-1 and phenolic-acids in AcOEtE-2, several of them with known anti-inflammatory properties.
The catechol mixture obtained from the PEE also shows inhibition of both enzymes isoforms, being more active against 5-hLOX.
The relevance of the previously detailed results, together with the fact that the AE of fresh leaves of L. caustica, showed good inhibitory activity against 5-hLOX, point in the direction of design new studies to validate in vivo, the traditional use of the decoction of L. caustica leaves and stems in Mapuche folk medicine, for treatment of joint inflammatory diseases and scaly skin lesions.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
AMR performed experiments, collected and analyzed data and contributed to the writing of the manuscript. CMC, AU and JE conceived the ideas. CMC contributed with the design and reagents for the biological assays. AB contributed with the LC-ESI-MS/MS analysis. AU and JE contributed to the writing of the manuscript and design of the research. All authors approved the final version of the manuscript.